Antioxidant and Anti-Obesity Potential

South African Journal of Botany 120 (2019) 104–111

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South African Journal of Botany

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Metabolite proﬁling and biological properties of aerial parts from Leopoldia comosa (L.) Parl.: Antioxidant and anti-obesity potential

M. Marrelli a,F.Aranitib, G. Statti a, F. Conforti a,⁎ a Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, I-87036 Rende, CS, Italy b Department of AGRARIA, University “Mediterranea” of Reggio Calabria, I-89100 Reggio Calabria, Italy article info abstract

Article history: Different anti-obesity drugs have proved unsuccessful due to their adverse effects. As a consequence, there is a Received 4 October 2017 growing interest in herbal remedies, with the aim to find new well-tolerated effective drugs. Leopoldia comosa Received in revised form 19 December 2017 (L.) Parl. grows in Central and Southern Europe, Northern Africa and Central and South-Western Asia, and the Accepted 6 January 2018 bulbs have been commonly used for food throughout history. In this study the effectiveness of L. comosa (L.) Available online 12 February 2018 Parl. leaves and inflorescences hydroalcoholic extracts and fractions was verified through the evaluation of pan- fi Keywords: creatic lipase inhibitory activity. The metabolite pro ling and the antioxidant activity were also investigated. fl – Antioxidant Chemical composition of L. comosa leaves and in orescences was assessed by means of GC MS and HPTLC anal- Chemical composition yses. The ethyl acetate fraction of leaves sample showed the best antioxidant activity, tested through DPPH and Leopoldia comosa β-carotene bleaching test. The effects on pancreatic lipase activity were assessed through the in vitro evaluation Lipase inhibition of the capacity to prevent p-nitrophenyl caprylate hydrolysis. Interestingly, leaves and inflorescences extracts and all their fractions were effective in inhibiting pancreatic lipase. The best anti-obesity potential was demon-

strated by the n-hexane and the ethyl acetate fractions of leaves sample, with IC50 values of 0.369 ± 0.020 and 0.336 ± 0.007 mg/mL. The same fractions of the inﬂorescences hydroalcoholic extract were also effective, with

IC50 values equal to 0.736 ± 0.045 and 0.780 ± 0.009 mg/mL. These results suggest that investigated samples could be a source of interesting compounds able to suppress dietary fat absorption. © 2018 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction few new drugs have been introduced, and the only two available in Europe are naltrexone sustained release (SR)/bupropion SR and Different drugs, such as dinitrophenol, amphetamines and liraglutide, recently approved in both USA and Europe (Krentz et al., sibutramine, have been utilized against obesity in the last decades. How- 2016). ever, despite some promising results, all of them have been withdrawn Recently, novel natural molecules have been studied and have been because of their serious adverse effects (Marrelli et al., 2016a). For many proved to play a role as key regulators in metabolic and inflammatory years, orlistat ((S)-2-formylamino-4-methyl-pentanoic acid (S)-1- pathways related to obesity, such as irisin (Ferrante et al., 2016a), [[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]-methyl]-dodecyl ester) was the apelin-13 (Ferrante et al., 2016b), endomorphin-2 (Brunetti et al., only anti-obesity drug approved in Europe for long-term use. This mol- 2013), chemerin (Brunetti et al., 2014a), and omentin-1 (Brunetti ecule is a chemically synthesized hydrogenated derivative of lipstatin, a et al., 2014b). natural product of Streptomyces toxytricini. Orlistat acts through the inhi- Nowadays, with the aim to find well-tolerated natural effective bition of gastric and pancreatic lipases, key enzymes for the digestion of drugs, there is a great interest towards herbal remedies (Marrelli dietary triglycerides that form unbound fatty acids from dietary triglyc- et al., 2016a). Different classes of phytochemicals have been recently erides that are absorbed at the brush border of the small intestine. Al- demonstrated to be effective against obesity, such as some polyphenols though orlistat is a reversible inhibitor of these two enzymes, it is (Meydani and Hasan, 2010), terpenes (Birari and Bhutani, 2007; Singh considered essentially irreversible, from a clinical point of view, because et al., 2015) and phytosterols (Trigueros et al., 2013). Saponins are of the slow reversibility of the binding (Lucas and Kaplan-Machlis, other interesting secondary metabolites with potential anti-obesity ac- 2001). However, also orlistat causes some side effects, such as diarrhea, tivity, able to play a role in body weight control by different mechanisms fecal incontinence and dyspepsia (Marrelli et al., 2016a). From 1998, just of action (Marrelli et al., 2016a). Herbal products can inhibit lipid absorption, increase energy expen- ⁎ Corresponding author. diture, decrease pre-adipocyte differentiation and proliferation. Other E-mail address: fi[email protected] (F. Conforti). anti-obesity mechanisms are a decreased lipogenesis or an increased

https://doi.org/10.1016/j.sajb.2018.01.006 0254-6299/© 2018 SAAB. Published by Elsevier B.V. All rights reserved. M. Marrelli et al. / South African Journal of Botany 120 (2019) 104–111 105 lipolysis (Hasani-Ranjbar et al., 2013). The potential health benefits of Table 1 different plant extracts have been demonstrated in the last few years Percentage yield of L. comosa (L.) Parl. leaves and inflorescences hydroalcoholic extracts and fractions. (Mocan et al., 2016, 2017; Savran et al., 2016) and some natural anti- obesity agents from medicinal plants have reached clinical trials Sample Fraction Yield %a (Marrelli et al., 2016a; Zengin et al., 2017). However, natural com- Leaves Raw extract 4.1 pounds have not been yet fully investigated and, potentially, new effec- n-Hexane 0.07 tive molecules could be discovered. CH2Cl2 0.17 As a continuation of our ongoing studies aimed to find effective anti- AcOEt 0.17 Inflorescences Raw extract 5.3 obesity botanicals (Conforti et al., 2012a; Marrelli et al., 2013, 2014, n-Hexane 0.07

2016b, 2016c), here we report the potential anti-obesity activity of CH2Cl2 0.05 Leopoldia comosa (L.) Parl. aerial parts. AcOEt 0.15 Leopoldia comosa (L.) Parl. (syn. Muscari comosum (L.) Miller, a % of extraction for 500 g of dried material. Data from one representative extraction. Liliaceae) grows in Central and Southern Europe, Northern Africa and Central and South-Western Asia. This species is present in the whole of Italy. The most common Italian popular name “cipollaccio” is due to 2.3. Total phenolic and flavonoid content estimation similarity of the bulb shape and taste to garlic, onion and leek. The bulb of L. comosa (L.) Parl. has been commonly used for food throughout The amount of total phenolics of L. comosa leaves and inflorescences history and, actually, it is commonly used as food in some rural commu- hydroalcoholic extracts was determined by the Folin-Ciocalteau nities in the south of Campania, in Apulia, in Basilicata and in Northern method (Araniti et al., 2014). Samples were mixed with distilled Calabria, where this species is just occasionally cultivated while the har- water, sodium carbonate solution and Folin-Ciocalteau reagent. After vesting of wild specimens is widespread. L. comosa bulbs are usually 2 h the absorbance of the blue color produced was measured at consumed fried in olive oil, occasionally mixed with cheese and eggs, 765 nm. Chlorogenic acid was used as standard and total phenolic con- boiled and served with a sweet and sour sauce, or pickled. Sometimes tent was expressed in mg per g of crude extract. the bulbs, after boiling with water and vinegar, are preserved with Flavonoid content was instead determined using a method based on olive oil (Casoria et al., 1999). The antioxidant activity of the bulbs of the formation of a flavonoid-aluminum complex (Marrelli et al., 2015). this plant has been already investigated (Pieroni et al., 2002). We also Each sample (2 mg/mL in EtOH 80%) was mixed with 2% AlCl3 in EtOH recently reported the phytochemical composition of this plant part, to- and absorbance was measured at 430 nm after 15 min. Results were gether with its interesting potential health benefits in the treatment of expressed as quercetin equivalents in mg per g of crude extract. obesity (Marrelli et al., 2017). Both quantifications were conducted in triplicate. Here we want to report the phytochemical content and the biological activity of L comosa aerial parts. Leaves and inflorescences were test- 2.4. HPTLC analysis ed for their ability to inhibit pancreatic lipase in vitro. Lipase inhibition is one of the most important strategies used by pharmaceutical industries For the analysis of polar compounds, the AcOEt fractions of L. comosa to decrease fat absorption after its ingestion (Marrelli et al., 2016a). The extracts were investigated by means of High Performance Thin Layer antioxidant activity and the phytochemical content of both leaves and Chromatography (HPTLC). One microliter of the two samples inflorescences were investigated as well. To the best of our knowledge, (50 mg/mL in methanol) was applied in triplicate on silica gel 60 glass this is the first study concerning the chemical composition and the bio- plates 20 cm × 10 cm (VWR International s.r.l., Milan, Italy) using logical properties of leaves and inflorescences of this plant species. Linomat 5 automated TLC applicator (CAMAG, Muttenz, Switzerland) according to the same operating conditions previously described 2. Materials and methods (Menichini et al., 2013). The presence of different flavonoids, such as quercetin, quercitrin, catechin, kaempferol, naringin and rutin was 2.1. Chemicals assessed using the corresponding standard at a concentration of 3 mg/mL. Plates were developed using the mobile phase AcOEt/ Ethanol, methanol, n-hexane, dichloromethane and ethyl acetate of CH Cl /CH COOH/HCOOH/H O (100:25:10:10:11; v/v/v/v/v) and suc- analytical grade were purchased from VWR International s.r.l. (Milan, 2 2 3 2 cessively dried at room temperature and at 100 °C for 10 min. TLC Italy). Lipase Type II, crude, from porcine pancreas, p-nitrophenyl plates were then observed under white light and at 254 and 366 nm caprylate (NPC), orlistat, 1,1-diphenyl-2-picrylhydrazyl (DPPH), before and after derivatization with NPR (1 g diphenylborinic acid ascorbic acid, β-carotene, linoleic acid, propyl gallate, Tween 20, Folin- aminoethylester in 200 mL of ethyl acetate) and anhysaldehyde Ciocalteu reagent, aluminum chloride, chlorogenic acid, quercetin and (1.5 mL p-anisaldehyde, 2.5 mL H SO ,1 mL AcOH in 37 mL EtOH). rutin were purchased from Sigma-Aldrich S.p.a. (Milan, Italy). All 2 4 HPTLC analyses allowed a good separation and visualization of the other reagents, of analytical grade, were Carlo Erba products (Milan, chemical constituents. Band stability was checked inspecting the Italy). resolved peaks at intervals of 12, 24 and 48 h, and repeatability was determined by running three analyses. RF values for main selected 2.2. Plant materials: collection and extraction procedure compounds varied less than 0.02%. The effects of small changes in the mobile phase composition and mobile phase volume were reduced by Leaves and inflorescences from wild L. comosa (L.) Parl. were collect- the direct comparison. ed in Calabria, Italy, in the Rende district (CS), at an altitude of 300 m, For rutin quantitative analysis a calibration curve (R2 = 0.9842) was 2013 (leg. F. Conforti, det. F. Conforti) in April 2013. Fresh plant material prepared using different concentrations of the standard compound (500 g of each sample) was extracted with 70% aqueous EtOH (3 L) (from 0.5 to 10.0 mg/mL) that were spotted on HPTLC plates to give dif- through maceration (48 h × 3 times) at room temperature. The resul- ferent amounts from 0.5 to 10 μg/band. tant total extracts were filtered and dried under reduced pressure to de- termine the weight and yield of extraction (Table 1). Obtained raw extracts were then suspended in methanol/water (9:1) and fractionated 2.5. GC–MS analysis with n-hexane. Remaining solution was suspended in water and por- tioned with dichloromethane and ethyl acetate. Percentage yields are Chemical composition of the n-hexane and dichloromethane reported in Table 1. fractions of leaves and inflorescences hydroalcoholic extracts was 106 M. Marrelli et al. / South African Journal of Botany 120 (2019) 104–111 investigated by gas chromatography–mass spectrometry (GC–MS). A scientific software, San Rafael, CA, USA). Significant differences among Hewlett-Packard 6890 gas chromatograph equipped with an SE-30 cap- means were analyzed using Tukey's post hoc test with P ≤ 0.05. illary column (100% dimethylpolysiloxane, 30 m length, 0.25 mm in di- To highlight metabolic differences between Leopoldia leaves and ameter, 0.25 μm film thickness) directly coupled to a selective mass inflorescences GC–MS data were analyzed using the software detector (model 5973, Hewlett Packard) was used. Electron impact ion- Metaboanalyst 3.0 (Xia et al., 2015). Data, expressed as metabolite con- ization was carried out in Electron Impact mode (EI, 70 eV). Analyses centrations, were checked for integrity and missing values were re- were realized using a programmed temperature from 60 to 280 °C placed with a small positive value. Data were successively normalized (rate 16° min−1) using helium as carrier gas. Injector and detector by the pre-added internal standard (fenchone at the concentration of were set at temperatures of 250° and 280 °C, respectively (Araniti 500 ppm), transformed through “Log normalization” and scaled et al., 2013). Identification of molecules was based on the comparison through Pareto-Scaling. Data were then classified through Principal of the GC retention factors with those of standards and the comparison Component Analysis (PCA) and metabolite variations were clusterized of the mass spectra with those present in the Wiley 138 library data of and presented through a heatmap (Araniti et al., 2017). Statistically sig- the GC–MS system. In order to highlight the metabolomic differences nificant differences between organs (leaves and inflorescences) were between leaves and inflorescences an internal standard (fenchone at showed through a volcano plot considering significantly different com- the concentration of 500 ppm) was added to the extracts. Repeatability pounds with a fold change ≥1.5 and a P value ≤ 0.05. The addition of the was determined by running three analyses. internal standard fenchone was carried out in order to both normalize the data, before the statistical analysis, and to do a relative quantifica- 2.6. Evaluation of antioxidant activity tion of the metabolite identified.

The antioxidant activity of L. comosa leaves and inflorescences extracts 3. Results and fractions was evaluated through the DPPH and the β-carotene bleaching test. The free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) 3.1. Extraction procedure was utilized for the determination of the radical scavenging potency of analyzed samples, as previously described (Conforti et al., 2012b). Test Fresh leaves and inflorescences wild from L. comosa (L.) Parl. were samples solutions at different concentrations (5–1000 μg/mL) were extracted with 70% aqueous EtOH through maceration procedure, and − added to a 10 4 M methanol solution of DPPH. Absorbance was mea- both raw extracts were then partitioned between n-hexane, dichloro- sured at 517 nm after 30 min in the dark using a Perkin Elmer Lambda methane and ethyl acetate. Flowers crude extract showed a higher 40 UV/VIS spectrophotometer. Experiments were run in triplicate and extraction yield than leaves sample (5.3% and 4.1%, respectively, ascorbic acid was used as positive control. Table 1). Yields of the different fractions obtained from the two extracts The β-carotene/linoleic acid system was instead used to assess the ranged from 0.05 to 0.17%. ability of the samples to inhibit lipid peroxidation (Menichini et al., 2013). A chloroformic 0.5 mg/mL β-Carotene solution (1 mL) was 3.2. Total phenolics and flavonoids content added to linoleic acid (0.02 mL) and 100% Tween 20 (0.2 mL). An emulsion was prepared after evaporation of chloroform and dilution Total phenolics and total flavoinoids contents were assessed by with 100 mL of water. Each sample (0.2 mL) was then added to 5 mL means of two spectrophotometric methods. Leaves and inflorescences of emulsion. Different concentrations of each sample were tested raw extracts showed an amount of phenolic compounds equal to (100, 50, 25, 10, 5, 1, 0.5 and 0.025 μg/mL). Obtained solutions were 50.50 ± 1.53 and 47.67 ± 2.62 mg/g of extracts, respectively, while a placed in a water bath at 45 °C and absorbance was measured flavonoid content of 4.59 ± 0.15 and 5.61 ± 0.07 mg/g was instead at 470 nm at initial time, 30 and 60 min. Experiments were run in trip- observed (Fig. 1). licate and propyl gallate was used as positive control. The antioxidant activity was measured in terms of successful prevention of β-carotene 3.3. HPTLC analysis bleaching. The identification of polar compounds was conducted by means of 2.7. Measurement of pancreatic lipase activity High Performance Thin Layer Chromatography (HPTLC). Analyses aimed to assess the presence of different flavonoids, such as quercetin, To assess the potential anti-obesity effects of L. comosa L. extracts and fractions, porcine pancreatic lipase (type II) activity was measured using p-nitrophenyl caprylate (NPC) as a substrate (Marrelli et al., 2013). Samples were incubated at 37 °C for 25 min with the enzyme solution (1 mg/mL in water), 5 mM NPC solution and Tris-HCl buffer (pH = 8.5). Absorbance was then measured at 412 nm. Experiments were run in triplicate and orlistat (final concentration 20 μg/mL) was used as positive control.

2.8. Statistical analysis

All measurements were carried out in triplicate and data were expressed as mean value ± S.E.M. Raw data were ﬁtted through non- linear regression in order to calculate the IC50 values (concentration providing 50% inhibition). Graphs were built using GraphPad Prism Software (version 6, San Diego, CA, USA).

Differences between IC50 values were evaluated through univariate analysis. Data were first checked for normality (D'Agostino-Pearson test) and tested for homogeneity of variances (Levene's test). Succes- Fig. 1. Total phenolic content (TPC) and total flavonoid content (TFC) of L. comosa (L.) fi sively, statistical signi cances among samples were assessed through Parl. leaves and inflorescences hydroalcoholic extracts. Data are expressed as means ± one-way analysis of variance (ANOVA) using SigmaStat Software (Jantel ES (n = 3) in mg/g of extract. M. Marrelli et al. / South African Journal of Botany 120 (2019) 104–111 107 quercitrin, catechin, kaempferol, naringin and rutin in the polar frac- Table 2 tions (AcOet samples) of the two hydroalcoholic extracts. The flavonoid Fatty acids and phytosterols from the n-hexane fractions of L. comosa (L.) Parl. leaves and inflorescences extracts. glycoside rutin was identified in both leaves and inflorescences samples, as illustrated in Fig. 2, which reports the chromatographic profiles of Fatty acidsa RTb mg/g of extractc AcOEt fractions from L. comosa extracts and the standard (Rf = 0.19). Leaves Inflorescences Quantitative analyses were conducted in order to compare the rutin Butanedioic acid 9.878 – Trd content of the two AcOEt fractions. Leaves of L. comosa showed a highest Caprylic acid 10.129 0.3 – content, with an amount of 0.118 ± 0.005 mg/g of crude extracts. A Azelaaldehydic acid 13.850 0.6 – value equal to 0.076 ± 0.001 mg/g was obtained for the inflorescences. Undecanoic acid 14.713 0.1 – Lauric acid 15.084 0.5 – Myristic acid 16.845 1.5 1.2 3.4. GC–MS analysis Oleic acid 17.302 – 0.1 Pentadecanoic acid 17.656 0.5 0.3 The GC–MS analysis carried out on L. comosa leaves and inflores- Tetradecanoic acid 17.702 0.2 – cences n-hexane fractions allowed the identification of 21 metabolites. Palmitic acid 18.588 19.8 15.5 In particular, 18 fatty acids and 3 phytosterols were identified Margaric acid 18.919 0.5 0.9 10,13-Octadecadienoic acid 19.445 0.2 – (Table 2). Palmitic acid was the most abundant compound, followed Linoleic acid 19.719 0.3 1.1 by stearic acid and myristic acid. Nine compounds were instead identi- Stearic acid 19.897 2.3 7.9 fied in the two dichloromethane fractions (Table 3). Different phenolic Linolenic acid 19.954 0.5 – acids, such as cinnamic acid, p-hydroxycinnamic acid, p-coumaric acid Eicosanoic acid 20.994 0.4 1.2 Behenic acid 22.263 0.9 – and ferulic acid were found in these two last samples. Lignocerid acid 23.743 0.2 0.6 Phytosterols(a) 3.5. Principal Component Analysis (PCA) 3α, 5-cyclo-ergosta-7,22-dien-6-one 28.224 – 0.3 Stigmast-7-en-3-ol, (3β,5α)- 34.699 0.3 – – The multivariate data analysis of the raw data, carried out through Stigmasta-3,5-dien-7-one 35.860 0.3 Principal Component Analysis (PCA), pointed out that the metabolic a Compounds listed in order of elution from SE30 MS column. b profile of leaves and inflorescences was clearly separated, confirming Retention time (as minutes). c Relative quantification based on the internal standard at known concentration added that the metabolic composition is extremely variable among different to the extract during the analysis. plant organs (Fig. 3-A). Leaves and inflorescences separation was d Compositional values less than 0.1 mg/g are denoted as traces. Data are expressed as achieved using the principal components PC1, which explained a vari- mean (n = 3). ance of 96.3%, and the PC2 component, which explained the lowest variance (1.8%) in the subspace perpendicular to PC1 (Fig. 3-A). The PCA loading plot reported in Fig. 3 highlighted that PC1 was mainly dominat- acid and linolenic acid, whereas the PC2 by 7-hydroxy-3-(1-1- ed by p-coumaric acid, syringic acid, mevalonic acid lactone, behenic dimethyl prop-2-nyl) coumarin, cinnamic acid, eicosanoic acid and margaric acid (Fig. 3-B). PCA and heatmap visualization of metabolomic data showed distinct segregation between L. comosa leaves and inflorescences (Fig. 3). Finally, data where analyzed through the volcano plot analysis considering statistically different compounds characterized by a fold change of 1.5 and a P value ≤ 0.05. The univariate analysis pointed out that 28 out of the 31 metabolites identified were significantly different among plant organs (Fig. 4 and Table 4). In particular, cinnamic acid, myristic acid and palmitic acid were the only compounds characterized by no statistical differences between leaves and inflorescences organs (Table 4).

Table 3 Chemical composition and relative quantiﬁcation of dichloromethane fractions from L. comosa (L.) Parl. leaves and inﬂorescences extracts.

Compounda RTb mg/g of extractc

Leaves Inﬂorescences

Mevalonic acid lactone 12.312 3.7 – Cinnamic acid 13.850 Trd tr Benzoicacid, 4-hydroxy-, methyl ester 14.187 2.2 tr Siringic aldehyde 16.090 – tr p-Hydroxycinnamic acid 16.867 0.9 tr p-Coumaric acid 17.228 – 3.3 Syringic acid 17.428 – 1.9 Ferulic acid 17.845 – tr 7-Hydroxy-3-(1,1-dimethylprop-2-enyl)coumarin 20.942 0.2 –

a Compounds listed in order of elution from SE30 MS column. b Retention time (as minutes). c Relative quantification based on the internal standard at known concentration added Fig. 2. HPTLC chromatographic profile of AcOEt fractions from L. comosa extracts. Mobile to the extract during the analysis. d phase: AcOEt/CH2Cl2/CH3COOH/HCOOH/H2O (100:25:10:10:11; v/v/v/v/v). A, AcOet Compositional values less than 0.1 mg/g are denoted as traces. Data are expressed as fraction of leaves extract. B, AcOet fraction of inflorescences extract. C, rutin (Rf = 0.19). mean (n = 3). 108 M. Marrelli et al. / South African Journal of Botany 120 (2019) 104–111

Fig. 3. PCA analysis carried on the metabolite identified and quantified in L. comosa leaves and inflorescences. Principal Component analysis model scores A) and loading plot B) of metabolite profile of leaves (Leaves_1 – Leaves_3, replicates of leaves samples) and inflorescences (Inflorescences_1 – Inflorescences_3, replicates of Inflorescences samples). Both score and loading plots were generated using the first two PCs, PC1 vs PC2, with the explained variances shown in brackets; C) overlay heat map of metabolite profiles identified in L. comosa organs (leaves and inflorescences). Each square represents the metabolite concentration expressed as false-color scale. Red or green regions indicate increase or decrease metabolite content, respectively.

3.6. Antioxidant activity

Radical scavenging activity of hydroalcoholic extracts and their fraction was assessed through the DPPH test. Testing different concen-

tration of samples, IC50 values equal to 154.8 ± 10.6 and 316.6 ± 11.69 μg/mL were obtained for leaves and inﬂorescences raw extracts, respectively (Table 5). The best activity was detected for the two polar fractions, particularly the ethyl acetate fraction of leaves sample

(IC50 =86.09±1.98μg/mL, Tukey's test). This same sample showed also the strongest capacity to protect linoleic acid from peroxidation, as assessed by the β-carotene

bleaching test (Table 5). After 30 min of incubation, an IC50 value of 23.73 ± 1.11 μg/mL was observed. The antioxidant activity, however, decreased after 60 min. Also inflorescences AcOEt fraction in- Fig. 4. Volcano Plot carried on the metabolite identified and quantified in L. comosa leaves and inflorescences. Statistically significant features, with a fold change ≥1.5 and a P value ≤ duced a good inhibitory activity, with an IC50 value equal to 53.35 ± 0.05, are reported in pink color (n = 3). 2.92 μg/mL. M. Marrelli et al. / South African Journal of Botany 120 (2019) 104–111 109

Table 4 Volcano plot data regarding the metabolites statically different between L. comosa leaves and inﬂorescences organs (data from Fig. 4).

Compounds FC log2(FC) P value -Log10(P) Azelaaldehydic acid 60.333 59.149 2.69E-04 75.709 Syringic acid 0.005172 −75.949 3.26E-04 74.871 p-Coumaric acid 0.002655 −85.571 9.03E-04 70.445 Mevalonic acid lactone 426.67 8.737 1.95E-03 67.099 Lauric acid 56.333 58.159 2.90E-03 65.383 Caprylic acid 29.333 48.745 2.04E-02 56.904 Benzoic acid. 4-hydroxy-. methyl ester 27.5 47.814 3.19E-02 54.961 3-α-5-Cyclo-ergosta-7-22-dien-6-one 0.025 −53.219 1.57E-01 48.035 Linolenic acid 70 61.293 1.58E-01 48.023 Tetradecanoic acid 26.667 4.737 2.00E-01 46.987 Undecanoic acid 12.667 3.663 2.90E-01 45.376 Behenic acid 100 66.439 6.86E-01 41.634 3β-5α-Stigmast-7-en-3-ol 45 54.919 6.89E-01 41.619 Rutin 1.553 0,63508 7.39E-01 41.311 10-13-Octadecadienoic acid 30 49.069 7.52E-01 4.124 Stigmasta-3-5-dien-7-one 0.01875 −5.737 0.000115 39.406 Linoleic acid 0.1907 −23.906 0.000299 35.241 Butanedioic acid 0.075 −3.737 0.000394 34.041 Siringic aldehyde 0.1875 −2.415 0.000538 32.694 Stearic acid 0.28936 −17.891 0.000697 3.157 p-Hydroxycinnamic acid 16.842 4.074 0.000998 30.009 Ferulic acid 0.17647 −25.025 0.001166 29.333 Oleic acid 0.033333 −49.069 0.002518 2.599 Lignocerid acid 0.33913 −15.601 0.003587 24.453 Pentadecanoic acid 20.513 10.365 0.007084 21.497 Margaric acid 0.41389 −12.727 0.008218 20.852 Eicosanoic acid 0.30652 −17.059 0.012591 18.999 7-Hydroxy-3-(1-1-dimethyl prop-2-nyl) coumarin 17.333 41.155 0.048782 13.117

Statistically significant differences between samples were observed, and 0.780 ± 0.009 mg/mL for the n-hexane and the ethyl acetate as biological activity of leaves fractions was significantly higher than the samples, respectively (Table 6). A lower but still interesting activity corresponding one of inflorescences. was demonstrated by the two dichloromethane fractions. Any significant difference was observed between the lipase inhibitory activity of

these two samples, with IC50 values of 1.409 ± 0.033 and 1.570 ± 3.7. In vitro inhibition of pancreatic lipase 0.027 mg/mL for leaves and inﬂorescences, respectively.

A very interesting inhibitory activity on pancreatic lipase was assessed for all tested samples (Table 6, Fig. 5). Leaves hydroalcoholic 4. Discussion extract (IC50 = 3.819 ± 0.119 mg/mL) was more active than inflorescence sample (IC50 = 6.561 ± 0.167 mg/mL), as evidenced by statistical Obesity is a serious health problem as this condition is associated analysis. The best anti-obesity potential was demonstrated by the with an increased risk of several diseases, including type II diabetes, n-hexane and the AcOet fractions of both samples, and particularly, cancer and cardiovascular events. Different drugs have already proved also in this case, leaves samples, with IC50 values of 0.369 ± 0.020 unsuccessful because of their serious adverse effects. Even Orlistat, nal- and 0.336 ± 0.007 mg/mL. The same fractions of the inflorescences trexone sustained release (SR)/bupropion SR and liraglutide, the only hydroalcoholic extract were also effective, even if the activity was lesser anti-obesity medications currently approved in Europe, cause some than that of the previous ones, with IC50 values equal to 0.736 ± 0.045 adverse effects. For this reason, there is a growing interest in herbal remedies, aiming to find well-tolerated natural effective drugs (Marrelli et al., 2016a). Moreover, herbal products have been proved Table 5 to be useful in the treatment of clinical symptoms related to obesity, Antioxidant activity of L. comosa (L.) Parl. leaves and inflorescences extracts and fractions.

Sample Fraction IC50 (μg/mL) DPPH test β-Carotene bleaching test Table 6 Inhibition of pancreatic lipase induced by L. comosa (L.) Parl. leaves and inflorescences ex- 30 min 60 min tracts and fractions. Leaves Raw extract 154.8 ± 10.6c 44.46 ± 2.64c N100 Sample Fraction IC (mg/mL) n-Hexane N1000 N100 N100 50 e CH2Cl2 N1000 N100 N100 Leaves Raw extract 3.819 ± 0.119 AcOEt 86.09 ± 1.98b 23.73 ± 1.11b 84.08 ± 3.60d n-Hexane 0.369 ± 0.020b d d d Inflorescences Raw extract 316.6 ± 11.69 84.42 ± 2.42 N100 CH2Cl2 1.409 ± 0.033 n-Hexane N1000 N100 N100 AcOEt 0.336 ± 0.007b e f CH2Cl2 472.1 ± 24.67 N100 N100 Inflorescences Raw extract 6.561 ± 0.167 AcOEt 102.4 ± 4.91c 53.35 ± 2.92c N100 n-Hexane 0.736 ± 0.045c ⁎ a d Ascorbic acid 2.00 ± 0.01 –– CH2Cl2 1.570 ± 0.027 ⁎ a a c Propyl gallate – 1.00 ± 0.02 1.00 ± 0.02 AcOEt 0.780 ± 0.009 ⁎ a Orlistat 0.018 ± 0.001 Data are expressed as mean ± SE (n = 3). Different letters along column (DPPH test), or between columns (β-carotene bleaching test) indicate statistically significant differences Data are expressed as mean ± SE (n = 3). Different letters along column indicate statisti- at p b 0.05 (Tukey's test). cally significant differences at p b 0.05 (Tukey's test). ⁎ Positive controls. ⁎ Positive control. 110 M. Marrelli et al. / South African Journal of Botany 120 (2019) 104–111

Fig. 5. Dose depending lipase inhibitory activity of leaves (A) and inflorescences (B) raw extracts and fractions. such as metabolic syndrome, oxidative stress and inflammation The pancreatic lipase inhibitory activity observed for the AcOEt frac- (Menghini et al., 2010, 2016; Tabatabaei-Malazy et al., 2015). To the tions could be in part related to the presence of the flavonoid glycoside best of our knowledge, this is the first study concerning L. comosa (L.) rutin, detected in both leaves and inflorescences ethyl acetate samples. Parl. aerial parts. The anti-obesity potential of leaves and inflorescences As a matter of fact, the interesting anti-obesity potential of this com- was evaluated testing their ability to inhibit pancreatic lipase activity pound (IC50 = 0.10 ± 0.01 mg/mL) has been demonstrated in our pre- in vitro. Both hydroalcoholic extracts and all their fractions were vious work (Marrelli et al., 2016b). effective. The metabolic profiling of leaves and inflorescences was also inves- 5. Conclusions tigated. Obtained raw extracts were fractionated using solvents with in- creasing polarity such as n-hexane, dichloromethane and ethyl acetate, To the best of our knowledge, this is the first study dealing with the and the chemical composition of obtained fractions was analyzed by potential health benefits of aerial parts of L. comosa (L.) Parl. Crude ex- means of GC–MS and HPTLC. Obtained data were then analyzed through tracts and fractions obtained from leaves and inflorescences induced Principal Component Analysis (PCA), that pointed out a clear separation an interesting dose-depending inhibition of pancreatic lipase in vitro. between the metabolic profile of leaves and inflorescences. In particular, It is worthwhile to further investigate these samples for their potential the univariate analysis pointed out that cinnamic acid, myristic acid and pharmacological effect in anti-obesity treatment. More studies are palmitic acid were the only compounds characterized by no statistical needed to correlate the activity of the two n-hexane samples to specific differences between leaves and inflorescences organs, while the other chemical constituents, with the aim to identify new lipid-lowering identified metabolites were significantly different. agent to be used as safer anti-obesity drugs. The biological activity of the bulbs of L. comosa has been already investigated. Pieroni et al. (2002) reported the free radical scavenging ac- Conflicts of interest tivity and the ability to inhibit bovine brain lipid peroxidation of the hydroalcoholic extract of this plant part. In a previous work (Marrelli The authors do not have any conflicts of interest. et al., 2017), we also compared the antioxidant potential of wild and cultivated bulbs, underlining that extract from wild bulbs showed References higher DPPH radical scavenging activity than extract obtained from cultivated ones. 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